This invention is in the field of cyclic polymerase-mediated reactions such as PCR. More specifically, this invention relates to altering the time within which the steps of such reactions are carried out. The methods of this invention are particularly relevant to reactions such as PCR as performed in devices that allow very short cycle times, such as microfluidic devices.
PCR and other cyclic polymerase-mediated reactions are standard tools of modern biological research, and are also commonly used for numerous applications including medical diagnostic procedures and forensic applications. PCR is based on three discrete, multiply repeated steps: denaturation of a DNA template, annealing of a primer to the denatured DNA template, and extension of the primer with a polymerase to create a nucleic acid complementary to the template. The conditions under which these steps are performed are well established in the art.
Generally, standard PCR protocols teach the use of a small number of cycles (e.g. 20-35 cycles) which are optimized for maximum efficiency in each cycle, i.e. to ensure that a highest possible percentage of template molecules is copied in each cycle. Typically, this entails cycle times of 1.2, or more minutes. For example, the standard reference Innis et al., PCR Potocols, A Guide to Methods and Applications (Academic Press, Inc.; 1990)(xe2x80x9cInnisxe2x80x9d) suggests the following conditions under the heading xe2x80x9cStandard PCR Amplification Protocolxe2x80x9d (at page 4):
Perform 25 to 35 cycles using the following temperature profile:
Such times, or longer, are typical in the field. Similar protocols can be found in, e.g. Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2d Edition), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (xe2x80x9cSambrookxe2x80x9d), which teaches a 6 minute cycle, and Ausubel et al., eds. (1996) Current Protocols in Molecular Biology, Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc. (xe2x80x9cAusubelxe2x80x9d), which teaches a 5 minute cycle. Accordingly, only up to about 20 or 35 cycles are typically required to generate a detectable and/or isolatable amount of product.
Recently, attempts have been made to shorten the time required for each cycle of a PCR. Such methods often reduce the time by, for example, performing the PCR in devices that allow rapid temperature changes, thereby eliminating much of the time previously required for PCR to xe2x80x9crampxe2x80x9d the temperature of the solution from one stage of the PCR to the next. In addition, it has been recognized that the use of apparatus that allow greater heat transfer, e.g. thin-walled tubes, turbulent air-based machines, also allow the use of shorter cycle times. For example, the RapidCycler(trademark), from Idaho Technologies, Inc. allows relative rapid ramping times between each temperature of a PCR and relatively efficient thermal transfer from the cycler to the samples. Accordingly, the Idaho Technologies Internet site (www.idahotec.com) provides an example of a PCR, wherein 30 cycles were completed in slightly less than 10 minutes.
Another example was discussed by Kopp et al. (1998) Science, 280:1046. Kopp et al. describe a microfluidic continuous flow PCR system where the PCR reactants were flowed through a chip having three discrete temperature zones. A channel was fixed within the chip to allow a fluid within the channel to pass through each of the zones repeatedly, generating a PCR comprising 20 cycles. By varying the speed by which the fluids flowed through the channel, Kopp et al. created a series of PCRs, each with cycles of varying lengths. Because of the design of this system, the reagents within the channel underwent essentially instantaneous changes in temperature. Thus, the cycle time in this system reflected the time at each temperature, with no substantial temporal contribution from the ramping times. Kopp et al. performed a series of 8 reactions, with cycle times varying from 60 to 4.5 seconds.
Consistent with previous studies, the shorter cycles used by Kopp et al. resulted in a significantly decreased amount of product. For example, a cycle time of about 12 seconds generated only about 45% of the product generated by a PCR using a 56 second cycle. A cycle time of 6.6 seconds generated less than about 10% of the 56-second cycle product. A cycle time of 4.5 seconds did not yield any detectable product.
None of these examples have challenged the teaching, well known to those of skill in the art, that regardless of the duration of the cycle, it is desirable to maximize the efficiency of the cycle. Accordingly, even those applications that suggest a low cycle time invariably suggest a standard, low number of cycles. For example, the system used by Kopp et al. was limited to 20 cycles, regardless of the length of the cycle. Similarly, the RapidCycler specifications page suggests using 30 cycle reactions. According to Kary Mullis, the Nobel Prize winning inventor of PCR (as quoted in Innis, supra), xe2x80x9cIf you have to go more than 40 cycles to amplify a single-copy gene, there is something seriously wrong with your PCR.xe2x80x9d
This invention is based, in part, on the surprising discovery that it is often desirable to perform PCR using short inefficient cycles. Specifically, despite their relative inefficiency, when short, inefficient cycles are repeated an unconventionally high number of times, it is possible to generate more product in the same amount of time or in less time than under standard conditions.
This invention is based on the surprising discovery that cyclic polymerase-mediated reactions, such as PCR, can be effectively carried out using very short cycles. As described herein, such reactions can be productively carried out even when the cycles are truncated to the point where they are significantly less efficient than under standard conditions. In particular, this invention demonstrates that performing a cyclic polymerase-mediated reaction using a higher than standard number of such short, inefficient cycles yields a high amount of product. In many cases, the amount of product generated using a high number of short cycles is greater than the amount generated in the same overall amount of time using standard conditions.
This invention teaches methods for performing cyclic polymerase-mediated reactions, wherein template molecules, polymerase enzymes, and primer molecules are incubated so as to extend the primer molecules, thereby duplicating at least a fraction of the template molecules. In these methods, the time allowed for the denaturation of the template and/or extension of the primer is less than under standard conditions, which are generally designed for optimum efficiency, i.e. maximum duplication of the template molecules. Consequently, when performed according to the methods described herein, each cycle of such a reaction is significantly less efficient than is typically accomplished using standard techniques in the art. In preferred embodiments, the percentage of template molecules that are duplicated in the short cycles steps is e.g. 90%, 70%, 50%, 30%, 10%, 5%, or less. Such cycles may be as short as 8-10, 6, 5, 4.5, 4, 2, 1, 0.5 seconds or less.
Because of the relative inefficiency of such short extension steps, the steps are repeated more times than is generally taught according to standard PCR protocols. In certain embodiments, the reaction comprises 30, 50, 70, 100, 200, 400, 1000, or more cycles.
In particularly preferred embodiments of this invention, cyclic polymerase-mediated reactions are performed using a high number of short, inefficient extension steps e.g. in a microfluidic device.
In certain embodiments of this invention, these processes are accomplished by changing the temperature of the solution containing the templates, primers, and polymerase. In such embodiments, the denaturation step is typically accomplished by shifting the temperature of the solution to a temperature sufficiently high to denature the template. In some embodiments, the hybridization step and the extension step are performed at different temperatures. In other embodiments, however, the hybridization and extension steps are performed concurrently, at a single temperature.
In some embodiments, the cyclic polymerase-mediated reaction is performed at a single temperature, and the different processes are accomplished by changing non-thermal properties of the reaction. For example, the denaturation step can be accomplished by incubating the template molecules with a basic solution or other denaturing solution.
Typically, the reactions described herein are repeated until a detectable amount of product is generated. Often, such detectable amounts of product are between about 10 ng and about 100 ng, although larger quantities e.g. 200 ng, 500 ng, 1 mg or more can also, of course, be detected. In many cases, however, a smaller amount of product is produced, for example, if a detection system is used that can detect less than 10 ng of product. In other cases, a larger amount of product is generated, for example if the product is to be isolated and purified for a separate, product-intensive application. In terms of concentration, the amount of detectable product can be from about 0.01 pmol, 0.1 pmol, 1 pmol, 10 pmol, or more.
The templates used in this invention can be derived from any of a number of different sources, including humans, mammals, vertebrates, insects, bacteria, fungi, plants, and viruses. Often, the templates are about 20-50, 50-100, 100-150, 175, 300, 500, 1000 nucleotides or longer.
The reactions described herein can be used to detect the presence or absence of a template from a sample of interest. Such methods of detection can be used, for example, for diagnostic or forensic purposes. In preferred embodiments, a plurality of samples, each potentially containing a template molecule or molecule, are subjected to the reactions of this invention, in parallel or in series, in order to screen the plurality of samples for the presence or absence of the template.
In certain embodiments, a nucleotide analog is incorporated into the product during one or more cycles of a cyclic polymerase-mediated reaction. Such an analog can be a detectable nucleotide, allowing the detection of the product during or following the reaction using visual or other means. Often, the nucleotide analog allows the sequencing of the product upon its isolation. For example, the analog can be a dideoxynucleotide (or other chain terminating residue) or a boronated nuclease-resistant nucleotide.
This invention also provides apparatus in which to perform the reactions described herein. Such apparatus are generally microfluidic devices. Such devices optionally include elements such as thermal control elements, detection zones, one or more source of test samples, and one or more receptacles for isolating the products of the reactions. In one embodiment, the apparatus is designed to alter the temperature of one or more fluids within the apparatus by joule heating, where the current relative to a cross dimension of a channel is controlled. Such apparatus are optionally part of an integrated system, which can include a computer that controls and/or monitors one or more of the above elements, which stores data, or which selects reaction conditions.
Kits for performing the reactions described herein, in certain cases including the above-described apparatus and integrated systems, instructional materials for practicing the methods herein, and/or packing materials are also provided.